Abstract

The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B(12) ). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) enzyme that synthesizes the unique α-linked nucleotide N(1) -(5-phospho-α-D-ribosyl)-DMB (α-ribazole-5'-P, α-RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α-RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α-ribazole (α-R) transporter and an α-R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α-R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α-R in a Salmonella enterica cobT strain. LinCblT transported α-R across the cell membrane, but not α-RP or DMB. UV-visible spectroscopy and mass spectrometry data identified α-RP as the product of the ATP-dependent α-R kinase activity of LinCblS. Bioinformatics analyses suggest that α-R salvaging occurs in important Gram-positive human pathogens.

Aerobic growth of L. innocua DD680 in MLM containing ethanolamine (100 mM). Optical density at 650 nm was measured for 12 h at 37°C. Corrinoids were added at 15 nM, α-R was added at 500 nM, and NH4Cl was added at 1 g l−1. Growth curves were obtained using an ELx808 Ultra Microplate reader (Bio-Tek Instruments). Each growth curve was performed in triplicate. Error bars of one standard deviation are indicated.